Characterization of Hit Compounds that Inhibit Glucocorticoid Receptor Translocation in a Cell-Based High Content Screen

[摘要] The goal of these studies was to characterize hit compounds identified in a high-content screen of over 220,000 compounds in a glucocorticoid receptor (GR) translocation assay. Hit compounds were those compounds that reproducibly inhibited the dexamethasone-induced translocation of GR from the cytoplasm to the nucleus in a concentration dependent manner. The translocation of GR occurs after glucocorticosteroid binds the ligand binding domain of GR maintained in a high affinity binding state by ATP bound hsp90 in a hetero-complex that includes hsp70, hsp40, Hop, and p23.. After glucocorticosteroid is bound to GR, the GR attaches to cytoplasmic dynein and is transported from the cytoplasm to the nucleus. Because the translocation of GR from the cytoplasm to the nucleus depends on the activity of both hsp90 and cytoplasmic dynein it is possible that a sub-set of hit compounds from the HCS might be hsp90 or cytoplasmic dynein inhibitors. The focus of this study was shifted to identifying hsp90 inhibitors after failure to show proof of concept that the previously published cytoplasmic dynein inhibitors ciliobrevin A and D could inhibit GR translocation in the GR translocation assay. After verification that hsp90 inhibitors do inhibit GR translocation in the GR translocation assay, the hit compound list was reduced from approximately 112 compounds down to five compounds that were presumed likely to bind hsp90 based on computational analyses. An ATPase activity assay was developed to test these five hit compounds against hsp90 and two other ATPases, myosin and kinesin; both of which are cytoskeletal motor proteins. Once these enzymes are optimized in the ATPase assay, they can be tested with the selected hit compounds.

[发布机构] the University of Pittsburgh